Cytomegalovirus (CMV) is a member of the human herpesvirus family, infecting between 50-100% of all individuals worldwide depending on age and socio-economic status.
CMV is naturally transmitted via saliva, urine or breast milk but can also be recovered from other body secretions. In addition, CMV can be transmitted transplacentally to the foetus, by geno-urinary contact during birth or intercourse, by blood transfusion (esp. white cells) and bone marrow cq. organ transplant.
After primary infection CMV persists in the body for the lifetime of its host in a state of dynamic latency, well controlled by the host immune system, and may be recovered periodically from different sites and body secretions.
Although generally benign, CMV infections can be devastating and fatal in individuals with immune defects, such as transplant recipients, AIDS patients, patients with genetically determined immunodeficiencies and newborns with an immature immune system.
Therapeutic intervention is possible using drugs affecting viral DNA replication but this is associated with significant toxicity. Modulation of host immunity is another effective way of controlling CMV infection, which can be achieved by vaccination, passive immunotherapy using immunoglobulins or T-cells, or by manipulation of immunosuppressive therapy.
Identification and monitoring of active (symptomatic) infection due to CMV cannot be done based upon clinical parameters alone, as these are diverse and variable, or by manipulation of immunosuppressive therapy.
Identification and monitoring of active (symptomatic) infection due to CMV cannot be done based upon clinical parameters alone, as these are diverse and variable, but heavily depends on rapid and accurate laboratory diagnosis.
CMV-specific diagnosis can be achieved by a variety of techniques directly detecting viral components or indirectly measuring changes in the hosts immune status. Reliable diagnostic approaches require sensitive and reproducible technology based upon well defined and highly CMV-specific reagents and a detailed understanding of the molecular processes underlying CMV-infection in the human host.
CMV is the largest and most complex of the human herpesvirinae, with a genome of 230-270 kilobases.
Characteristic for a herpesvirus, the structural components of the CMV virion include a protein-DNA core, an icosahedral capsid consisting of 162 capsomer subunits, an amorphous layer of proteins called tegument and a surrounding proteo-lipid envelope essential for virus infectivity.
Although in vivo CMV can be found in a variety of host cells and tissues, with different levels of viral gene expression, efficient gene expression and viral DNA replication in vitro is only possible in primary fibroblast cells of human origin. Therefore this system has been most widely used to study viral gene expression, replication and characterization of related protein products.
Following binding, de-envelopment and penetration into a susceptible host cell (e.g. human fibroblast), tegument proteins from the incoming virion activate the expression of viral genes of the immediate early (IEA) class, encoding a limited number of protein species with broad-acting transcriptional activating functions, capable of transactivating the transcription of a wide variety of viral and host genes. Viral genes activated by IEA products predominantly consist of enzymes involved in nucleotide metabolism and DNA synthesis and are classified as the group of early antigens (EA). In the last stage of infection and essentially depending on the synthesis of new viral DNA templates by the virally encoded DNA polymerase (an EA component), a third (late antigen (LA)) group of viral proteins is expressed, consisting of the structural components required for assembly and release of viral progeny.
Both in vivo and in vitro, this sequence of gene expression can be interrupted at various stages leading to non-lytic (abortive or defective) infection cq. persistence, which is characterized by restricted gene expression patterns. These patterns are both dependent on the type of cell infected and on its activation and differentiation status. Viral gene products thus expressed still may alter host cell characteristics resulting in aberrant behavior and function.
In addition to the expression of its own genes in a 3-step cascade regulated fashion, CMV stimulates host cell gene expression following infection, thus complicating the specific analysis and purification of viral products.
In blood, CMV can be found in sporadic monocyte/macrophages (replicating), lymphocytes (abortive/restricted), polymorph nuclear cells (only PP65 protein) and circulating endothelial cells (replicating). Furthermore CMV can be detected in smooth muscle cells lining the arteries and smaller blood vessels (restricted/lytic) and fibroblast and macrophage-like cells in a variety of tissues.
In the absence of effective immune responses CMV can spread to virtually any tissue and cell type in the body.
The presence of infectious virus in the blood as determined by culture techniques is considered the best marker associated with active symptomatic infection, whereas the presence of viral DNA is a marker for virus carrier status.
CMV is associated with a wide variety of disease syndromes both in the immunocompetent and in the immunocompromised host, although the latter are much more frequent and associated with significantly greater morbidity and mortality.
Primary infection in the immunocompetent host usually go unnoticed. However CMV is considered to be causing 10% of the mononucleosis syndrome in adolescents and young adults and is frequently associated with acute nonA-G hepatitis. Primary infection in pregnant women is associated with the transplacental transfer of CMV to the foetus.
CMV is the leading cause of birth defects in the world, infecting between 0.5 and 3% of all newborns. In about 10% of infected newborns CMV-related defects can be detected, which are most serious in case of a primary infection in the mother.
CMV is named the “Troll of transplantation: as it is the most frequent infectious cause of complications in solid organ and bone marrow transplant recipients.
CMV also is a leading cause of disease in HIV positives and AIDS patients, most frequently associated with pneumonia, retinitis and gastrointestinal complications.
Finally, CMV frequently causes disease in patients with genetically determined immunodeficiencies, cancer patients and patients with autoimmune diseases.
The quality of the host immune status determines the balance between CMV replication and spread and viral persistence in a dormant latency state.
Host immune responses are induced and maintained upon encounter of newly synthesized or stable persisting viral gene products expressed during the different stages of infection at various sites/tissues in the body.
Depending on the virus-host interactions at the molecular level—which may be different for each individual—immune responses are built up gradually following primary infection until a state of balance is achieved, which is called latency.
Determination of the quality and quantity of host immune responses to CMV are of diagnostic and prognostic importance and have been widely used to determine immune status to CMV, to establish the donor/recipient CMV carrier status in the transplant setting and to identify/monitor acute infections in a variety of CMV-associated disease syndromes.
Diagnostic assays may directly measure the presence and quantity of virus in body fluids, by means of virus culture or DNA detection/quantitation techniques. However, only CMV detection in the blood is considered as a reliable parameter for diagnosis of active infection in man, as the virus may be secreted intermittently in urine and saliva in most healthy CMV carriers.
DNA detection in blood is not considered a reliable predictor for active CMV infection as latently infected CMV DNA positive leukocytes can be detected in most if not all healthy carriers, depending on the sensitivity of the assay used.
Measurement of CMV RNA expression in blood leukocytes may be useful for detecting active virus replication in the blood depending on the genomic target sequence analyzed.
Alternatively the antigenaemia assay, i.e. the quantitation of blood leukocytes (esp. polymorph nuclear cells) for the presence of intracellular PP65(UL83), has proven to be a reliable diagnostic parameter correlating well with active and symptomatic CMV infection in a variety of CMV disease syndromes.
In contrast to methods for the direct detection of virus or viral products, the analysis of humoral immune responses to CMV can be used as an indirect reflection of CMV activity in the human host. CMV serology is probably the most widely used approach for the diagnosis and monitoring of active CMV infections and for determination of CMV carrier status. A wide variety of techniques are available which are both rapid and cheap for detecting IgM, IgG or IgA antibodies to CMV.
Whereas the detection of CMV IgM and to a lesser extent CMV IgA is directly indicative for (recent) active infection, especially during a primary infection, the measurement of CMV IgG may be applied both for determination of CMV carrier status and for diagnosing/monitoring CMV disease.
During primary infection IgG antibodies are formed rapidly and remain present for life. Besides the presence of CMV IgM a significant rise in CMV IgG is reflecting active infection in the host. The latter may be complicated in case of full blood transfusion or immunoglobulin therapy.
Besides being a measure of active infection the generation of an antibody response to CMV is a reflection of immunocompetence and can be used to guide antiviral therapy in the immunocompromised. Detection of a brisk antibody response during antiviral therapy may be used as a sign to end the therapy and to allow the immune system to take over the control of CMV in the host.
Thus, serology is a reliable and versatile tool in the diagnosis and monitoring of CMV infections in the human host.
In the design of serological assays a crucial ingredient is the CMV-specific antigen preparation which serves to bind immunoglobulins in the sample to be analyzed. Thus it is important to define the fine molecular specificity of anti-CMV antibody responses in order to allow standardization. Such studies have been initiated in recent years but have proven difficult due to the diversity of anti-CMV antibody responses and the complexity of the CMV system.
Although the 235KB CMV genome of prototype strain AD169 has been sequenced completely and over 200 potential protein encoding open reading frames have been identified, only some 40 of these proteins have been biochemically and immunologically studied to date. A limited number of CMV polypeptides have been defined as targets for human antibody responses, such as pp150 (UL32), pp72(UL122/123), pp65(UL83), pp52(UL44), pp38(UL80), pp28(UL99), gB(UL55) and MDBP(UL57). Additional immunoreactive polypeptides have been defined by their molecular weight in CMV-infected cell extracts analyzed using the immunoblot technique, but their coding frames on the CMV genome have not been defined yet.
As stated above many different techniques are currently used for CMV serology.
These methods either use cell culture derived (semi-purified) antigen extracts, partially purified extracellular vinons and dense bodies or more defined (recombinant) proteins and fragments thereof. Due to this variation of methodology and lack of uniform and well defined reagents, CMV serology currently is not well standardized. Reliable diagnosis and further standardization of CMV serology therefore calls for the use of molecular defined and highly purified CMV antigens.
Such antigens may be produced and purified from CMV cell culture, but this is expensive and complicated by the presence of a multitude of host cell proteins. Recombinant CMV proteins expressed in alternative host systems require even higher grade of purification as patient sera may have antibodies to such host cell components leading to potential false positive results.
An example of the use of recombinant proteins can be found in Patent Application WO 95/00073, wherein a mixture of recombinant antigens (fusion proteins) are used to detect CMV specific IgM.
Synthetic peptides represent a highly defined alternative for such protein antigens and can be produced and purified in a highly reproducible manner.